Hepatitis E virus, known previously to cause much acute viral hepatitis in developing countries, has recently been identified as a cause of sporadic hepatitis in industrialized countries. It also has been found to cause chronic hepatitis in severely immune-compromised persons such as those receiving organ transplants. Inability to propagate the virus efficiently in cell culture or in small animal models has inhibited attempts to characterize its molecular biology or identify its pathogenic determinants. Two genotypes of the virus infect humans only, but the remaining two genotypes that infect humans are zoonotic and swine are a known reservoir. It seems certain that other animal reservoirs for human HEV must exist but they have yet to be identified and the biological factors that permit some strains to cross species boundaries are completely unknown. In FY2010 we used transfected human hepatoma cells and polarized CaCo-2 intestinal cells to study HEV release from infected cells . We showed that virus egress in cell culture depends on the small viral ORF3 protein and requires an intact PXXP motif which is thought to be necessary for interaction with host-cell signaling proteins. We found that ORF3 protein is localized to the apical membrane in polarized cells but that this localization is not dependent on the PXXP motif . Intrahepatic transfection of rhesus monkeys with mutated RNA transcripts demonstrated that successful infection requires an intact PXXP motif. In FY2010 we isolated a new strain of genotype 3 HEV from the stool of a chronically infected patient. This high tittered virus stock was able to infect swine cells and cells from other animal species in addition to human cells. This represents the first demonstration of HEV infection of non-primate cells in vitro. The discovery that this strain displayed a much higher infectivity titer on swine cells than on human cells indicates that this virus/cell culture set will be the first useful in vitro system suitable for studying cross-species infection of HEV as it relates to zoonotic infections. We also serially passaged this new virus strain in cultured human hepatoma cells and adapted it to grow more efficiently in these cells. During the adaptation process we discovered that a recombinant HEV with a 174 nucleotide insertion as well as numerous point mutations was selected. We have constructed infectious cDNA clones of the first passage (without insert) and 5th passage (with insert) virus and will use them to determine which mutations are important for efficient growth in cell culture and for species tropism. We will introduce mutations into our cDNA clones until optimal growth is achieved and we will use these clones to analyze the functions and interactions of the viral proteins : some of these studies will be guided by results of microarray studies recently performed on liver tissue from chimpanzees experimentally infected with HEV. Prior to FY2010 we had unsuccessfully attempted to recover HEV sequence from rats that were ELISA-positive for antibodies to human HEV capsid protein. In FY2010 ,we used primers published by a German group and were finally able to obtain sequence from our laboratory rats experimentally infected with serum from wild rats. The rat sequence is only distantly related to that of human HEV so we have constructed an expression plasmid to synthesize what we predict is the rat neutralization peptide and we plan to use it to develop a more sensitive and specific ELISA for antibodies to the rat virus.